2.1. Cell Culture and Experimental Treatments

SH-SY5Y cells transfected with wild type APP (695 isoform) and BACE1 (SH-SY5Y-APP/BACE1, maximum number of passages = 26) were cultured at 37 °C in 5% CO₂ with Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), 100 units/mL penicillin, and 260 μg/mL Zeocin. Cells were treated with 1 μM insulin (Human Recombinant Zinc, Gibco, #12585014) for 2 h. Cells were incubated with 10 μM Akti (Akt inhibitor, Calbiochem, #124018) or 50 μM OSMI-1 (OGT inhibitor, Sigma-Aldrich, #SML1621) for 2 h. Cells were incubated with 50 μM PUGNAc (OGA inhibitor, Tocris Bioscience, Bristol, UK, #3384) or 10 μM Thiamet G (OGA inhibitor, Sigma, Burlington, MA, USA, #SML0244) for 24 h. SH-SY5Y-APP/BACE1 cells were provided by T.-W. Kim (Columbia University Medical Center) [60].